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目的探讨B细胞特异的Moloney鼠白血病病毒插入位点1(Bmi-1)基因沉默对细胞增殖与侵袭的影响及可能的分子机制。方法收集经病理确诊的乳腺癌及癌旁组织,采用实时荧光定量PCR检测Bmi-1的表达差异;采用LipofectamineRNAi MAX转染试剂将靶向Bmi-1基因的小干扰RNA(siRNA)转染MCF-7细胞,流式细胞术检测Bmi-1沉默后细胞周期和凋亡的改变,Western blot法检测凋亡相关基因P21、Bax、Bcl-2的表达变化。TranswellTM侵袭实验检测MCF-7细胞侵袭力变化,Western blot法检测上皮钙黏素(E-cadherin),神经钙黏素(N-cadherin)、波形蛋白(vimentin)的表达水平。结果乳腺癌组织Bmi-1 mRNA表达量高于癌旁组织,沉默Bmi-1基因可使MCF-7细胞周期阻滞于G1期,凋亡细胞增多;与空白对照组、阴性对照siRNA转染组相比,Bmi-1-siRNA组中P21与Bax的表达水平明显上调,Bcl-2明显下调。沉默Bmi-1基因表达可抑制MCF-7细胞的侵袭力,与对照组相比,下调Bmi-1可增强E-cadherin的表达,减少N-cadherin、vimentin的表达。结论下调Bmi-1的表达可引起MCF-7细胞G1期阻滞,促进细胞凋亡,与P21表达上调,Bax/Bcl-2比率升高有关;下调Bmi-1水平可抑制MCF-7细胞侵袭性,与抑制肿瘤细胞上皮间质转化有关。
Objective To investigate the effect and possible molecular mechanism of B cell-specific Moloney murine leukemia virus insertion site 1 (Bmi-1) gene silencing on cell proliferation and invasion. Methods Bmi-1 expression was detected by real-time fluorescence quantitative polymerase chain reaction (PCR). The siRNA targeting small interfering RNA (siRNA) of Bmi-1 gene was transfected by Lipofectamine RNAi MAX transfection reagent The changes of cell cycle and apoptosis after Bmi-1 silencing were detected by flow cytometry. The expressions of apoptosis-related genes P21, Bax and Bcl-2 were detected by Western blot. The invasiveness of MCF-7 cells was detected by TranswellTM invasion assay. The expression of E-cadherin, N-cadherin and vimentin were detected by Western blot. Results The expression of Bmi-1 mRNA in breast cancer tissues was higher than that in paracancerous tissues. The silencing of Bmi-1 gene blocked the cell cycle arrest of G1 phase and increased the number of apoptotic cells in MCF-7 cells. Compared with the blank control group and the negative control siRNA transfection group Compared with the Bmi-1-siRNA group, P21 and Bax expression levels were significantly increased, Bcl-2 was significantly down-regulated. Silencing Bmi-1 gene expression can inhibit the invasiveness of MCF-7 cells. Compared with the control group, down-regulation of Bmi-1 can enhance the expression of E-cadherin and decrease the expression of N-cadherin and vimentin. Conclusions The down-regulation of Bmi-1 can induce G1 arrest and promote apoptosis in MCF-7 cells, which is related to the up-regulation of P21 and the increase of Bax / Bcl-2 ratio. Down-regulation of Bmi-1 can inhibit the invasion of MCF-7 cells Sex, and inhibit tumor cell epithelial-mesenchymal transformation.